Histones are protein subunits of the nucleosome which in eucaryotes package DNA into chromatin. Among the four core histone protein families, the histone H2A family is unique in that it includes three evolutionarily conserved subfamilies, suggesting that nucleosomes containing these different H2A species may have different functions. Our long range goal is to elucidate what these functions may be, with particular emphasis on the H2AZ and H2AX subfamilies. During this year we have been following up on some insightful results on the possible roles of these histone subfamilies in chromatin. Since our discovery of the connection in cells and animals between phosphorylation of serine-139 of histone H2AX and exposure to ionizing radiation, we have extended this connection to include apoptosis, substantiating the involvement of DNA double-strand breaks (dsbs). Working with Dr. Yves Pommier's group, we found that histone H2AX became phosphorylated on serine-139 in several cell lines undergoing apoptosis. Cell line-inducer pairs that were studied included HL60-staurosporine, HL60-EGTA, Jurkat-staurosporine, Jurkat-antifas, and MCF7/ADR-UNC01. In Jurkat cells incubated with anti-fas antibody, DNA dsbs and phosphorylated H2AX were detected about the same time as annexin V binding. ZVAD which inhibits DNA fragmentation during apoptosis also inhibited H2AX phosphorylation in the cell line-inducer pairs studied. These results show that H2AX phosphorylation is an early feature of apoptosis and that apoptotic cells may respond to DNA breaks similarly to cells exposed to ionizing radiation. Last year we reported that the fraction of H2AX (PO4-Ser139) increased with the amount of ionizing radiation. In various cell lines, about 1% of the H2AX is converted to H2AX (PO4-Ser139) for each Gy of ionizing radiation at doses below 50 Gy. We have extended these results to non- lethal amounts of ionizing radiation. In SF268 cells, H2AX (PO4-Ser139) was detected after 1.2 and 3.6 Gy, amounts of radiation that allow 50% and 5% survival respectively. In mice H2AX (PO4-Ser139) was detected in liver after the non-lethal dose of 6 Gy. In these cases the amount of H2AX (PO4-Ser139) formed per Gy was similar to that found after lethal doses. H2AX (PO4-Ser139) is found in significantly lower amounts in the M059J human tumor line which is deficient in DNA dsb repair and DNA-activated protein kinase. However, in further studies with rodent cell lines deficient in DNA dsb repair, H2AX (PO4-Ser139) formation was found to be similar to control cell lines. These results suggest that DNA- activated protein kinase may not be the enzyme responsible for H2AX (PO4-Ser139) formation. However, H2AX (PO4-Ser139) formation may still be involved in DNA dsb repair upstream of this enzyme. Recombinant human H2A histones are being used in various structural and functional assays. We have obtained evidence that the H2AZ-H2B dimer is unstable at physiological salt conditions compared to the H2AX-H2B and H2A1-H2B dimers. We are also progressing on preparing homologous cell knockouts of H2AX and H2AZ.